The objective of this design-driven proposal is to optimize and employ scaffold-free co-cultures for the generation of biomimetic fibrocartilages for the repair or replacement of the temporomandibular joint meniscus and the mandibular cartilage surfaces. Using co-cultures of fibrochondrocytes and articular chondrocytes, we recently generated large constructs of clinically relevant dimensions that were fibrocartilage-like in appearance and composed of extracellular matrix (ECM) suggestive of fibrocartilage. Moreover, constructs similar to native tissue were achieved with fibrochondrocytes. Motivated by these findings, it is our hypothesis that the co- cultures can be optimized using bioactive agents and mechanical stimuli to form biomimetic fibrocartilage constructs. To address this hypothesis, we propose the following specific aims: 1) to optimize the use of chondroitinase ABC and the growth factors transforming growth factor -1 (TGF-1) and insulin-like growth factor 1 (IGF-I) using serum-containing medium or chemically defined medium in the scaffold-free co-culture of fibrocartilage; 2) to enhance the biomimetic fibrocartilage constructs with mechanical stimulation; and 3) to examine synergistic effects of bioactive agents and mechanical stimuli. Chondroitinase ABC has been found in preliminary studies to significantly increase construct tensile properties. Our group has also demonstrated that TGF-1 and IGF-I significantly increase ECM production in fibrocartilage constructs. The use of hydrostatic pressure and direct compression stimulation has been shown by our laboratory to have beneficial effects on articular cartilage and fibrocartilage constructs, and we will apply these stimuli individually and in combination to further enhance the constructs. Constructs will be examined histologically for glycosaminoglycan (GAG) and collagen, and immunohistochemically for collagen I and II. GAG, collagen, and DNA content will be quantified, followed by ELISA to measure collagen I and II. Biomechanical evaluation will include compression, tension, and creep indentation testing. Furthermore, microarray analysis will be used to study potential synergisms that may arise in the use of the exogenous stimuli. Finally, the engineered constructs will be implanted in the nude mouse to examine viability and stability.